Neuroprotective liposome compositions and methods for treatment of stroke

ABSTRACT

Methods for the treatment of stroke, such as stroke of undetermined origin, by administration of xenon (Xe)-loaded liposome compositions are provided. In some aspects, Xe is encapsulated in echogenic liposomes and release of Xe can be enhanced by application of ultrasound stimulation. Compositions for use in treating stroke, such as liposomes loaded with Xe or Xe in combination with H2 or H2S, are also provided.

This application claims the benefit of U.S. Provisional PatentApplication No. 61/682,130, filed Aug. 10, 2012, the entirety of whichis incorporated herein by reference, and the benefit of Chinese PatentApplication no. 201210356929.9, filed Sep. 21, 2012.

The invention was made with government support under Grant Nos.NS067454, HL 074002 and HL 059586 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of medicine,biochemistry and molecular biology. More particularly, it concernscompositions and methods of using biologically protective liposomes forthe treatment of stroke.

2. Description of Related Art

Thrombotic and hemorrhagic strokes, also known as cerebrovascularaccidents (CVA), are, together, the fourth leading cause of death in theUnited States and the most common cause of adult disabilities. Bothtypes of stroke are characterized by a rapid loss of brain function dueto disturbance in the blood supply to the brain. In thrombotic stroke,occlusion of a cerebral artery caused by a blood clot, results in braintissue ischemia or obstruction of cerebral blood flow to a portion ofthe brain, and ultimately brain damage. Conversely, in hemorrhagicstroke, blood leaks or bursts from broken blood vessels inside or on thesurface of the brain leading to neurological damage. Regardless of thetype of stroke, early protection of brain tissues against the acutevascular events caused by thrombosis or hemorrhage by medicalintervention remains the most crucial element to save patients' lives.The early brain tissue protection can broaden the safe window fordifferential diagnosis and effective treatment. Despite similar initialsymptoms at onset, effective therapeutic intervention depends upon thetype of stroke that a patient is experiencing. For example,administration of tissue plasminogen activator (tPA) has proven at leastpartially effective in treatment of thrombotic stroke, but is counterindicated for treatment of hemorrhagic stroke. There remains a need fornew effective therapeutics for treatment of stroke, in particular,therapeutics that are amenable to immediate administration upon strokeonset.

SUMMARY OF THE INVENTION

In a first embodiment there is provided a method for treating a strokein a subject comprising administering an effective amount of acomposition comprising Xenon-loaded echogenic liposomes (Xe-ELIP) to thesubject. In some aspects, the subject has been determined to have ahemorrhagic stroke. In a further embodiment, there is provided a methodtreating a stroke of undetermined origin in a subject (i.e., a subjectnot yet determined to have a thrombotic or hemorrhagic stroke)comprising administering an effective amount of a composition comprisingXe-ELIP to the subject. Thus, in some aspects, a method is provided fortreating both hemorrhagic and thrombotic stroke in a subject (e.g., asubject that has been diagnosed with a hemorrhagic and/or thromboticstroke). In still further aspects, a method of the embodiments isdefined as a method of treating an intracranial aneurysm or subarachnoidhemorrhage in a subject comprising administering an effective amount ofa composition comprising an effective amount of Xe-ELIP to the subject.In some embodiments there is provided a composition comprising Xe-ELIPin a pharmaceutically acceptable carrier.

In a further embodiment methods and compositions of the embodimentsemploy a different noble gas loaded in echogenic liposomes (in place ofor in addition to Xenon). For example, in some aspects, echogenicliposomes for use according to the embodiments are loaded with Xenon,Helium, Argon, Krypton, Neon or a mixture thereof. Thus, in someaspects, there is provided a method for treating a stroke in a subjectcomprising administering an effective amount of a composition comprisingXenon, Helium, Argon, Krypton, or Neon-loaded echogenic liposomes to thesubject. In further aspects, the subject has been determined to have ahemorrhagic stroke. In a further embodiment, there is provided a methodtreating a stroke of undetermined origin in a subject (i.e., a subjectnot yet determined to have a thrombotic or hemorrhagic stroke)comprising administering an effective amount of a composition comprisingXenon, Helium, Argon, Krypton, or Neon-loaded echogenic liposomes to thesubject. Thus, in some aspects, a method is provided for treating bothhemorrhagic and thrombotic stroke in a subject (e.g., a subject that hasbeen diagnosed with a hemorrhagic and/or thrombotic stroke). In stillfurther aspects, a method of the embodiments is defined as a method oftreating an intracranial aneurysm or subarachnoid hemorrhage in asubject comprising administering an effective amount of a compositioncomprising an effective amount of Xenon, Helium, Argon, Krypton, orNeon-loaded echogenic liposomes to the subject. In some embodimentsthere is provided a composition comprising Xenon, Helium, Argon,Krypton, or Neon-loaded echogenic liposomes in a pharmaceuticallyacceptable carrier.

In a further embodiment there is provided a method of treating athrombotic stroke in a subject comprising (a) administering an effectiveamount of a first composition comprising Xe-ELIP to the subject; and (b)administering an effective amount of a second composition comprisingtissue plasminogen activator (tPA) to the subject. For example, incertain aspects, Xe-ELIP and tPA are administered sequentially oressentially simultaneously (e.g., in a composition comprising Xe-ELIPand tPA). In some aspects, a method of the embodiments comprisesadministering the second composition about or less than about 2, 3, 4,5, 6, 7, or 8 hours after administration of the first composition. Instill further aspects, administration of the first composition is withinabout 6 hours or less of stroke onset. In yet still further aspects, thesecond composition further comprises and effective amount of Xe-ELIP. Instill a further aspect, a method of the embodiments comprises (a)administering an effective amount of a first composition comprisingXe-ELIP to a subject having a stroke or symptoms of a stroke ofundetermined origin; (b) identifying whether the subject is sufferingfrom a thrombotic or hemorrhagic stroke; and (c) administering aneffective amount of a second composition comprising tPA to a subjectidentified as having a thrombotic stroke.

Thus, in further embodiment, there is provided a pharmaceuticalcomposition comprising Xe-ELIP and tPA. In some aspects, the tPA of thecomposition is comprised in liposomes (e.g., echogenic liposomes). Infurther aspects, the composition comprises a slurry of at least twodifferent liposome where the first liposomes are comprised of Xe-ELIPand the second liposomes comprise tPA (e.g., wherein tPA liposomes areessentially free of Xe).

In yet a further embodiment there is provided a pharmaceuticalcomposition comprising noble gas-loaded-ELIP (e.g., Xe-ELIP) andliposomes (e.g., echogenic liposomes) loaded with a further biologicallyactive gas component. For example, in some aspects noble gas-loadedechogenic liposomes may further comprise (or may be administered inconjunction with liposomes comprising) H₂S and/or H₂. Likewise, in someaspects, noble gas-loaded echogenic liposomes may further comprise (ormay be administered in conjunction with liposomes comprising) nitrousoxide and/or nitric oxide. For example, in some aspects, the gases arecomprised in separate liposomes in the composition (e.g., a slurry ofliposomes loaded with different gases). In further aspects, two or moreof the gases are comprised in the same liposomes. For example, acomposition can comprise liposomes that comprise Xe and NO, Xe and N₂Oor Xe, NO and N₂O. In another example, a composition can compriseliposomes that comprise Xe and H₂, Xe and H₂S or Xe, H₂ and H₂S. Infurther aspects, liposomes of the embodiments comprise between about0.1% and 5%, 0.1% and 3% or 0.5% and 2% H₂S (as a percent of total gasin the liposome). For example, the liposomes can comprise about 1% H₂Sand about 99% Xe. In yet further aspects, liposomes of the embodimentscomprise between about 1% and 50%, 5% and 40% or 10% and 40% H₂ (as apercent of total gas in the liposome). For example, the liposomes cancomprise about 30% H₂S and about 70% Xe.

Certain aspects of the embodiments concern compositions comprising tPA.In some aspects, the tPA is purified or recombinant mammalian tPA (e.g.,human tPA). Such tPA compositions may be comprised in a pharmaceuticallyacceptable carrier. In certain aspects, the tPA is comprised inliposomes. Liposomes for use in encapsulating tPA can be selected fromany other those known in art or detailed herein. For instance, in someaspects, the tPA is comprised in echogenic liposomes of the embodiments.In certain preferred aspects, liposomes comprising tPA are essentiallyfree of Xe (i.e., tPA and Xe are not loaded into the same liposomevesicle).

Compositions in accordance with the embodiments can be administered to asubject via an array of methods. For example, in some aspects,compositions (e.g., Xe- or tPA-containing compositions) are administeredintravenously, intra-arterially, intracranially, via intravenousinfusion or via intra-arterial infusion. In preferred aspects,compositions of the embodiments are administered shortly after the onsetof a stroke or stroke symptoms, such as about or less than about 1, 2,3, 4, 5, 6 or 8 hours of stroke onset or the onset of stroke symptoms.Thus, in some aspects, compositions of the embodiments are administeredby a first responder (e.g., a nurse or medical technician).

In certain aspects, a method of the embodiments comprises administeringone or more doses of an ELIP composition (e.g., a Xe-ELIP composition)to a subject. For example, in some aspects, a subject is administered adose of between about 0.6 and 3.0 mg/kg of Xe-ELIP, such as a dose ofbetween about 0.8 and 2.8 mg/kg (mg-lipid/kg-subject). In furtheraspects, a subject is administered a dose of between about 1.0 and 2.5mg/kg of an ELIP composition, such as about 1.14 mg/kg or about 2.27mg/kg. In still further aspects, ELIP compositions are provided thatcomprise a single unit dosage (e.g., of Xe-ELIP) in a suitablecontainment means. For example, the single unit dosage (i.e., a dosesuitable for a human subject of about 60 kg) can be about 100 to about3,000 mg; about 250 to about 2,000 mg; about 250 to about 1,000 mg; orabout 400 to about 900 mg (e.g., 480-900 mg) of ELIP with anencapsulated gas (based on total lipid weight). In still furtheraspects, a single unit dosage of Xe-ELIP can be defined by the totalvolume of encapsulated Xe. In some aspects, the total volume of Xe in asingle dose is less than about 5 ml, such as between about 0.1 and 2.5ml; about 0.2 and 2.0 ml; or 0.5 and 1.5 ml (e.g., a dose of about 1.0ml) of Xe. Thus, in some specific aspects, a single unit dosage ofXe-ELIP is provided comprising between 250 to about 1,000 mg of lipidand between about 0.1 and 2.5 ml of Xe in suitable containment means. Askilled artisan will recognize that any of the forgoing dose ranges mayalso be applied to Xe-ELIP compositions that include H₂ and/or H₂S (inthis case gas volumes can be applied to the total amount of encapsulatedgas).

In still a further aspect, a composition (e.g., an ELIP composition) ofthe embodiments is administered to a subject in multiple doses. Forexample, in some aspects, the composition is administered to the subjecta second, third or fourth time. In certain aspects, the formulation,dosage or route of the second dosage or subsequent administration can beadjusted relative to the first administration. In some aspects, thesecond or subsequent administration is about or less than about 2, 3, 4,5, 6, 7, or 8 hours after the initial administration (e.g., within about4-6 hours after the first administration). In some aspects, acomposition of the embodiments is administered to a subject twice withinabout 12 hours of the onset of a stroke or stroke symptoms.

In preferred aspects a subject for treatment in accordance with theembodiments is a human in subject. However, in some aspects, the subjectcan be a non-human animal, such a non-human primate or a domesticatedanimal such a horse, dog or cat. In further aspects, the subject isidentified as having symptoms of stroke, such as sudden memory loss,full or partial paralysis, disorientation, trouble speaking, suddenvision impairment, numbness of a portion of the body, sudden, severe,headache or trouble walking or balancing. In some aspects, the subjecthaving a stroke or stroke symptoms has not been identified as having athrombotic stroke or a hemorrhagic stroke. In still further aspects, thesubject has been identified as having a thrombotic stroke and/or ahemorrhagic stroke.

In further aspects, administration of an ELIP composition of theembodiments to a subject further comprises applying ultrasoundstimulation to the subject in an amount effective to promote gas releasefrom the liposomes. For example, the ultrasound stimulation can beapplied after or concomitantly with administration of the ELIPcomposition. In some aspects, the ultrasound stimulation is appliedafter the administration of an ELIP composition (e.g., Xe-ELIP) such asabout or less than about 10 seconds, 20, seconds, 30 seconds or 1, 2, 3,4 or 5 minutes after the administration. In certain cases the ultrasoundstimulation is applied at or near the site of desired gas release by theliposomes. For example, in the case of a subject having a stroke,ultrasound stimulation can be applied to the head or neck (e.g., at thecarotid artery) thereby stimulating release of liposomal payloadproximal to the brain.

A variety of methods for applying ultrasound stimulation to a subjectare known and can be used in accordance with the embodiments. Forexample, the ultrasound stimulation can be applied with a conventionalultrasound probe or a cervical collar ultrasound device (e.g., toprovide stimulation at the neck). The power and frequency of ultrasoundstimulation applied to a subject can vary, but generally will be anamount effective to promote Xe release (e.g., in vivo release) fromliposomes. For example, the ultrasound stimulation can be applied at afrequency of between about 1 and 8 MHz, with a mechanical index ofbetween about 0.1 and 1.4.

In still further aspects a method of the embodiments further comprisesadministering at least a second therapeutic agent to the subject. Forexample, in the case of thrombotic stroke the second therapeutic can bea blood clot reducing thrombolytic agent. In further aspects, the secondtherapeutic agent is an anti-inflammatory agent or a neuroprotectiveagent. In some specific aspects, e.g., in the case of thrombotic stroke,tPA is administered to a subject.

In certain specific aspects, the second therapeutic agent comprises H₂Sand/or H₂-loaded echogenic liposomes. In some cases, the H₂S and/orH₂-loaded echogenic liposomes and the Xe-loaded echogenic liposomes arecomprised in the same composition. For example, a composition of theembodiments can comprise echogenic liposomes that, separately, compriseXe, H₂S and/or H₂. Alternatively, the compositions can compriseechogenic liposomes that comprise two or three gases (e.g., two or moregases selected from Xe, H₂S and H₂).

In still further aspects, a method of administration in accordance withthe embodiments comprises preparing a liquid liposome suspension priorto administration to a subject. For example, in some aspects, the liquidliposome suspension is prepared no more than 30 minutes, 10 minutes, 5minutes or 2 minutes prior to administration. For example, in someaspects, preparing a liquid liposome suspension comprises suspendinglyophilized liposomes in a solution or thawing a frozen liposomesuspension.

In further aspects an ELIP composition of the embodiments comprisesadditional components, such as preservatives, stabilizers and/or salts.In some aspects, ELIP compositions comprise at least a firstcryoprotectant. Cryoprotectants for use according to the embodimentsinclude, without limitation, mannitol, glycerol, trehalose,1,2-propanediol or dimethylsulfoxide (DMSO).

As used herein an echogenic liposome refers to a liposome that can beimaged by ultrasound. In particular aspects, an echogenic liposome is aliposome that comprises a gas component (e.g., Xe, H₂S and/or H₂), suchas gas comprised in the hydrophobic layer of the liposome. Echogenicliposome compositions and methods for making such composition areprovided for example is U.S. Pat. Nos. 5,612,057; 5,858,399; and7,976,743, each of which is incorporated herein by reference. In someaspects, liposomes of the embodiments (e.g., ELIP compositions) aredefined by the average particle size. For example, in some aspects theliposomes have an average size of about 0.4 to 10 microns or 0.8 to 10microns (e.g., an average size of about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 microns).

A wide array of components can be used to formulate a liposome of theembodiments, such as ELIP loaded with a gas such as Xe, H₂ and/or H₂S.For example, a liposome of the embodiments can comprise any form ofphosphatidylcholine (PC) (such as dipalmitoyl phosphatidylcholine(DPPC)), any form of phosphoethanolamine (PE), a polyethylene glycol(PEG) or a PEGylated phospholipid, any form of phosphatidylglycerol (PG)(such as 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG))and/or cholesterol. In some aspects, a liposome comprises at least onePC, PE, negatively-charged lipid, PEGylated lipid (e.g., PEG2000-DPPE)and cholesterol molecule. In some specific aspects, a liposome comprisesDPPC, Egg PC (EPC), PEG2000-DPPE, DPPG and cholesterol. In still furtheraspects a liposome of the embodiments consists of or consistsessentially of DPPC, EPC, PEG2000-DPPE, DPPG, cholesterol and Xenon. Instill further aspects, a liposome consists of or consists essentially ofDPPC, EPC, PEG2000-DPPE, DPPG, cholesterol, Xe and H₂, H₂S or acombination of H₂ and H₂S.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Characterization of xenon-containing liposome andultrasound-triggered xenon release. (a) Traditional liposome withoutgas. (b) Liposome containing Xe, made by the pressurized-freezingmethod, with Xe entrapped in the lipid bilayer as a dissolved gas or abubble. (c and d) Electron microscopic image of traditional liposome andgas-containing liposome showed a wide lipid bilayer for gas-containingliposomes. Intravascular ultrasound imaging of traditional liposome (e)and Xe-containing liposome (f) showed a high ultrasound reflectivityfrom Xe-containing liposomes. (g) Ultrasound release of xenon fromXe-ELIP has two phases: a fast release in first 30 min followed by aslow release lasting more than 18 h (half-life 4.97±0.7 hours).Ultrasound triggered the release of Xe from Xe-ELIP in a power-dependentmanner (h).

FIG. 2: Time window of Xe-ELIP's neuroprotective effect. Coronal brainsections (TTC staining) of middle cerebral artery occlusions withouttreatment (a) and with Xe-ELIP (7 mg/kg) treatment at 10 min (b), 1 h(c) and 3 h (d) after reperfusion. The white areas are the infarctedregions after middle cerebral artery occlusion; Quantification of theinfarct volume of the brain showed that Xe-ELIP administration at 10 minand 1 h after reperfusion was significantly different from the notreatment group. Neurological assessments of limb placement (f), beamwalking (g), and grid walking (h) showed similar results as the TTCstaining. Data are means± standard error.

FIG. 3: Effects of Xe-ELIP on BDNF expression and apoptosis. Westernblot analysis of BDNF (a), phos-Akt (b) and phos-ERK (c) in cerebralcortex tissue 24 h after stroke showed that Xe increased the expressionof BDNF (d), total Akt (e) and phos-ERK (f). TUNEL staining in thepenumbral region of brain sections from the sham-operated group (g),stroke group (h) and stroke with Xe-ELIP treatment group (i) showedreduction of apoptosis in Xe-ELIP-treated animals. The Western blots andphotomicrographs of apoptosis are representative of three independentexperiments. Data are means±SD.

FIG. 4: Dose response of Xe-ELIP's neuroprotective effect. Coronal brainsections (stained with TTC) of middle cerebral artery occlusions wereimaged without treatment (a), and with 3.5 mg/kg (b), 7 mg/kg (c) and 14mg/kg (d) of Xe-ELIP. The white areas are the infarcted regions aftermiddle cerebral artery occlusion. Quantification of the infarct volumeof the brain is shown in (e). Neurological assessments of limb placement(f), beam walking (g), and grid walking (h) are provided in theindicated graphs. Data are means±standard error.

FIG. 5: Effects of IV tPA in combination with Xe delivery on cerebralischemia. Representative TTC-stained coronal brain sections showingbrain infarction in rats 3 days after middle cerebral artery occlusionwere imaged after (a) sham, (b) ischemic stroke without treatment, (c)ischemic stroke with tPA treatment (d) ischemia stroke with Xe-ELIP incombination with tPA. (e) A comparison of infarct sizes betweentreatment groups showing a 69% reduction in infarct volume with tPAalone and a 75% reduction with Xe-ELIP combined with IV tPA.Neurological assessments of limb placement (f), beam walking (g) andgrid walking (h) are shown in the indicated graphs. Data aremeans±standard error.

FIG. 6 a-b: (a), Xe-ELIP provides neuroprotective effects whenadministered either before or after restoring blood flow. (b), Percentclot mass loss in animals after the indicated treatments with Xe-ELIPand/or tPA.

FIG. 7: Schematically shown is an example protocol for the preparationof echogenic liposomes of the embodiments.

FIG. 8: Results show that Xe-ELIP decreases the bleeding in a filamentperforation subarachnoid hemorrhage (SAH) rat model.

FIG. 9: Results show that Xe-ELIP improves the general neurologicalevaluation scales and motor function of SAH rats.

FIG. 10: Results show that Xe-ELIP prevents neuronal apoptotic celldeath. Representative photomicrographs show TUNEL staining of brainsections from hemorrhage stroke group (upper panels), and hemorrhagestroke with Xe-ELIP treatment group (bottom panels). The left panels areDAPI stain of total cells. Center panels are tunnel stain of apoptoticcells. Right panels are merged images.

FIG. 11: Results show that Xe-ELIP decreases the mortality rate of SAHrats but does not greatly affect brain edema and cerebral blood flow.

FIG. 12: Schematics show time lines for the experiments of Example 4.

FIG. 13: Results of stroke treatment with Xe-ELIP, H₂S/Xe-ELIP andH₂/Xe-ELIP in the tMCAO model are shown. (a) Representative TTC-stainedcoronal brain sections showing brain infarction. (b) A summarycomparison of infarct volumes between treatment groups. (c) Graphicalrepresentation of TUNEL staining of brain sections from each treatmentgroup.

FIG. 14: Graphs show the results of behavioral tests for neurologicdisability in rats treated with the indicated composition as assed bylimb placement, beam walking and grid walking, as indicated.

FIG. 15: Graphs show the results of the tests of efficacy of Xe-ELIPreagents in the protection of human brain astrocytes against the H₂O₂(10 mM) cytotoxicity using lactate dehydrogenase (LDH) release assays.

FIG. 16: Graphs show the results of the tests of Xe-ELIP cytotoxicity instem cells. H₂O₂ but not the Xe and Xe-ELIP reagents cause significantLDH release in murine embryonic stem cells. Cells were treated withXe-reagents (3 mg ELIP/30 μl gas/ml) and control medium for 4 hours, and2 hours before the end of cultures; 10 mM H₂O₂ was added.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The Present Invention

Stroke also known as cerebrovascular accident (CVA), is the thirdleading cause of death in the United States and the most common cause ofadult disabilities. A stroke is characterized by a rapid loss of brainfunction due to disturbance in the blood supply to the brain. Inthrombotic stroke, occlusion (a blood clot) of a cerebral artery,resulting in obstructed blood flow to a portion of the brain.Conversely, in hemorrhagic stroke blood leaks or bursts from bloodvessels in the brain leading to neurological damage. Early therapeuticand/or surgical intervention is crucial in mitigating neurologicaldamage from stroke. However, depending upon the type of stroke thetherapy administered is quite different, to the extent thattherapeutics, such as tPA, cannot be administered to a patent having astroke unless the type of stroke has been positively identified.Unfortunately, the most crucial factor in treatment of stroke is timelyintervention, which limits the usefulness of therapeutics such as tPA.

Studies detailed here demonstrate the synthesis of a new kind ofechogenic liposome that encapsulates Xenon gas (Xe-ELIP). The Xe-ELIPformulations are demonstrated to quickly and effectively releaseencapsulated Xe upon application of ultrasound (FIG. 1). Compositionscomprising these liposomes are also shown to be effective not only inthe treatment of thrombotic stroke, but also surprisingly, for treatmentof hemorrhagic stroke (see FIGS. 2, 4, 8, and 9). Thus, for the firsttime, Xe-ELIP represents a therapeutic that can be administered to apatient immediately following a stroke (or the onset of stroke symptoms)and before the type of stroke can be positively identified. Unlike anyother identified therapy Xe-ELIP thus, can protect neurons from theinsults that result from both blood clot (ischemia) and hemorrhage.Importantly, Xe-ELIP buys the patient crucial time by protecting thebrain from excessive neurological damage (as assessed both by actualneuronal damage and behavioral testing) while surgical or othertherapeutic interventions can be implemented. Accordingly, this newclass of therapeutic offers the possibility of significantly improvingclinical outcome of all classes of stroke.

Interestingly, studies detailed herein likewise demonstrate that Xe-ELIPcan work in concert with tPA administration in providing effectivetreatment for thrombotic stroke (see, FIG. 5). Here again, earlytreatment with Xe-ELIP prevents excessive neuronal damage while thestroke diagnosis is assessed. Subsequent administration of tPA (with orwithout additional Xe-ELIP) then mediates clot break-down, resulting insignificantly better clinical outcome as compared to tPA alone.

Further studies presented herein demonstrate the neuroprotective effectof Xe-ELIP can be yet further enhanced by co-administration (oradministration of co-encapsulated) H₂ or H₂S gas. As shown in FIGS.13-14 both H₂ and H₂S, when administered in conjunction with Xe-ELIP,are able to further reduce infarct volume and further improve outcome instoke subjects as assessed by behavioral testing. Furthermore, asdemonstrated in FIG. 16 none of the ELIP compositions (comprising Xe orXe and H₂ or H₂S) showed significant toxicity as assessed in murineembryonic stem cells. Accordingly, the further incorporation of H₂and/or H₂S into Xe-ELIP compositions may yet further enhance theirneuroprotective efficacy.

II. Liposomes and Liposome Compositions

A “liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes may be characterized as havingvesicular structures with a bilayer membrane, generally comprising aphospholipid, and an inner medium that generally comprises an aqueouscomposition. Liposomes provided herein include unilamellar liposomes,multilamellar liposomes and multivesicular liposomes. Liposomes providedherein may be composed of positively charged, negatively charged orneutral phospholipids. In certain embodiments, the liposomes are neutralin charge.

A multilamellar liposome has multiple lipid layers separated by aqueousmedium. They form spontaneously when lipids comprising phospholipids aresuspended in an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Lipophilic molecules or molecules with lipophilicregions may also dissolve in or associate with the lipid bilayer.

In specific aspects, a gas is capsulated in a liposome to generate anechogenic liposome that can be imaged and/or disrupted by theappropriate application of ultrasound. Specific methods for gasencapsulation are detailed below and exemplified in Example 1 and FIGS.1 and 7.

A liposome used according to the present embodiments can be made bydifferent methods, as would be known to one of ordinary skill in theart. For example, a phospholipid (Avanti Polar Lipids, Alabaster, Ala.),such as for example the neutral phospholipid dioleoylphosphatidylcholine(DOPC), Dipalmitoyl Phosphatidylcholine (DPPC) and/or EPC, can bedissolved in an alcohol or other organic solvent and then mixed with acomponent for inclusion in the lipid bilayer. The mixture may furtherinclude various detergents. Typically, a lipid mixture is vortexed,frozen in a dry ice/acetone bath and lyophilized overnight. Thelyophilized preparation is stored at −20° C. or less for extendedperiods of time. When required the lyophilized liposomes arereconstituted, for example, in 0.9% saline.

Alternatively, a liposome can be prepared by mixing lipids in a solventin a container, e.g., a glass, pear-shaped flask. The container shouldhave a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

In other alternative methods, liposomes can be prepared in accordancewith other known laboratory procedures (e.g., see Bangham et al., 1965;Gregoriadis, 1979; Deamer and Uster, 1983; Szoka and Papahadjopoulos,1978, each incorporated herein by reference in relevant part).Additional liposomes which may be useful with the present embodimentsinclude cationic liposomes, for example, as described in WO02/100435A1,U.S. Pat. No. 5,962,016, U.S. Application 2004/0208921, WO03/015757A1,WO04029213A2, U.S. Pat. No. 5,030,453, and U.S. Pat. No. 6,680,068, allof which are hereby incorporated by reference in their entirety withoutdisclaimer. A process of making liposomes is also described inWO04/002453A1. Neutral lipids can be incorporated into cationicliposomes (e.g., Farhood et al., 1995). Various neutral liposomes whichmay be used in certain embodiments are disclosed in U.S. Pat. No.5,855,911, which is incorporated herein by reference. These methodsdiffer in their respective abilities to entrap aqueous material andtheir respective aqueous space-to-lipid ratios.

The size of a liposome varies depending on the method of synthesis.Liposomes in the present embodiments can be a variety of sizes. Incertain embodiments, the liposomes are small, e.g., less than about 100nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less thanabout 50 nm in external diameter.

In preparing such liposomes, any protocol described herein, or as wouldbe known to one of ordinary skill in the art may be used. Additionalnon-limiting examples of preparing liposomes are described in U.S. Pat.Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505,and 4,921,706; International Applications PCT/US85/01161 andPCT/US89/05040; U.K. Patent Application GB 2193095 A; Mayer et al.,1986; Hope et al., 1985; Mayhew et al. 1987; Mayhew et al., 1984; Chenget al., 1987; and Liposome Technology, 1984, each incorporated herein byreference).

In certain embodiments, the lipid based nanoparticle is a neutralliposome (e.g., a DOPC liposome). “Neutral liposomes” or “non-chargedliposomes”, as used herein, are defined as liposomes having one or morelipid components that yield an essentially-neutral, net charge(substantially non-charged). By “essentially neutral” or “essentiallynon-charged”, it is meant that few, if any, lipid components within agiven population (e.g., a population of liposomes) include a charge thatis not canceled by an opposite charge of another component (i.e., fewerthan 10% of components include a non-canceled charge, more preferablyfewer than 5%, and most preferably fewer than 1%). In certainembodiments, neutral liposomes may include mostly lipids and/orphospholipids that are themselves neutral under physiological conditions(i.e., at about pH 7).

Liposomes and/or lipid-based nanoparticles of the present embodimentsmay comprise a phospholipid. In certain embodiments, a single kind ofphospholipid may be used in the creation of liposomes (e.g., aphospholipid, such as DPPC (composed of all saturatedphosphatidylglycerol or phosphatidylserine), may be used to generate aliposome). In other embodiments, more than one kind of phospholipid maybe used to create liposomes (e.g., DPPC and EPC).

Phospholipids include, for example, phosphatidylcholines,phosphatidylglycerols, and phosphatidylethanolamines; becausephosphatidylethanolamines and phosphatidyl cholines are non-chargedunder physiological conditions (i.e., at about pH 7), these compoundsmay be particularly useful for generating neutral liposomes. In certainembodiments, the phospholipid DOPC is used to produce non-chargedliposomes. In certain embodiments, a lipid that is not a phospholipid(e.g., a cholesterol) may be used

Phospholipids include glycerophospholipids and certain sphingolipids.Phospholipids include, but are not limited to,dioleoylphosphatidylycholine (“DOPC”), egg phosphatidylcholine (“EPC”),dilauryloylphosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine(“DMPC”), dipalmitoylphosphatidylcholine (“DPPC”),distearoylphosphatidylcholine (“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”), 1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”), 1-palmitoyl-2-stearoyl phosphatidylcholine(“PSPC”), 1-stearoyl-2-palmitoyl phosphatidylcholine (“SPPC”),dilauryloylphosphatidylglycerol (“DLPG”),dimyristoylphosphatidylglycerol (“DMPG”),dipalmitoylphosphatidylglycerol (“DPPG”), distearoylphosphatidylglycerol(“DSPG”), distearoyl sphingomyelin (“DSSP”),distearoylphophatidylethanolamine (“DSPE”), dioleoylphosphatidylglycerol(“DOPG”), dimyristoyl phosphatidic acid (“DMPA”), dipalmitoylphosphatidic acid (“DPPA”), dimyristoyl phosphatidylethanolamine(“DMPE”), dipalmitoyl phosphatidylethanolamine (“DPPE”), dimyristoylphosphatidylserine (“DMPS”), dipalmitoyl phosphatidylserine (“DPPS”),brain phosphatidylserine (“BPS”), brain sphingomyelin (“BSP”),dipalmitoyl sphingomyelin (“DPSP”), dimyristyl phosphatidylcholine(“DMPC”), 1,2-distearoyl-sn-glycero-3-phosphocholine (“DAPC”),1,2-diarachidoyl-sn-glycero-3-phosphocholine (“DBPC”),1,2-dieicosenoyl-sn-glycero-3-phosphocholine (“DEPC”),dioleoylphosphatidylethanolamine (“DOPE”), palmitoyloeoylphosphatidylcholine (“POPC”), palmitoyloeoyl phosphatidylethanolamine(“POPE”), lysophosphatidylcholine, lysophosphatidylethanolamine, anddilinoleoylphosphatidylcholine.

Phospholipids may be from natural or synthetic sources. Phospholipidsfrom natural sources, such as egg or soybean phosphatidylcholine, brainphosphatidic acid, brain or plant phosphatidylinositol, heartcardiolipin and plant or bacterial phosphatidylethanolamine (orhydrogenated versions thereof) are used, in certain embodiments, as thephosphatide. In some aspects, PEGylated lipids are employed such asPEG2000-DPPE=1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (which could be all mPEG Phospholipids or allPhosphatidylethanolamine).

It will likewise be understood by a skilled worker that the molar ratiosof various liposome components may be adjusted to optimize delivery,encapsulation, etc. In some aspects, for example, a liposome comprisesDPPC:EPC:PEG2000-DPPE:DPPG:CH in a ratio of about30-50:10-30:5-15:5-15:10-20, or about 40-50:20-30:5-10:5-10:10-20. Someexamples of specific ratios include, without limitation, 50:20:10:10:15;60:30:10:10:12; 46:23:8:8:15; 47:27:9:8:13; or 48:28:7:7:13.

In certain embodiments, the lipid-based vesicle is a DOTAP:cholesterolnanoparticle. DOTAP:cholesterol nanoparticles are prepared by mixing thecationic lipid DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)-propane)with cholesterol. Vesicles can further be prepared with a nucleic acidand can form a structure (called a “sandwich’) where the nucleic acidappears to be condensed between two lipid bilayers (U.S. Pat. Nos.6,770,291 and 6,413,544).

A. Gas-Loaded Liposomes

The present invention, in certain embodiments, provides methods for thefacile production of gas-containing liposomes with simultaneous drugencapsulation. In exemplary embodiments (see Example 1), liposomes ofphospholipid and cholesterol were prepared by conventional procedures ofhydrating the lipid film, sonicating, freezing and thawing. The lipidsgenerated contain air by including a step after sonication where thelipid is placed under pressure with the gas of interest. Afterequilibration, the sample is frozen. The pressure is then reduced toatmospheric and the suspension thawed. This procedure leads toentrapment of air in amounts up to about 10% by volume by lipiddispersions at moderate (10 mg/ml) concentrations. The amount of gasencapsulated increases with gas pressure and lipid concentration.Utilizing 0.32 M mannitol to provide an aqueous phase with physiologicalosmolarity, 1, 2, 4 or 6 atm of pressure was applied to 4 mg of lipid.This would led to encapsulation of 10, 15, 20, and 30 μl of gas,respectively. While the present embodiments are not limited to anyparticular mechanism, the mechanism for gas encapsulation presumablydepends upon the fact that air (predominantly nitrogen and oxygen), likemost solutes, dissolves poorly in ice and is excluded from the ice thatforms during freezing. The excluded air then comes out of solution asair pockets that are stabilized in some form by a lipid coating. Thepresence of air in these preparations sensitizes them to ultrasound suchthat up to half of their aqueous contents (which could include a watersoluble drug) can be released by short (e.g., 10 second) applications ofultrasound.

The present invention provides methods to introduce gas into liposomessuch that they not only reflect ultrasound, but also release theircontents when exposed to ultrasound or other triggering procedure. Ofpractical importance is that the method, which, in certain embodiments,uses elevated-pressure in combination with freezing, is very simple andallows ready encapsulation of solutes along with incorporation of a gasof choice. The method is suitable for the preparation of both anultrasound contrast agent and an ultrasound-controlled drug deliverysystem.

Conventional procedures for preparing liposomes do not allow forincorporation of a gas because the solubility of gas in water is low.According to Henry's Law, however, the solubility of a gas in a liquidis directly proportional to the pressure of that gas over the liquid. Asolution is regarded as undersaturated, saturated or supersaturated whenthe pressure of the gas is less than, equal to or larger than theequilibrium saturation value in local temperature. Thus, if the pressureis increased, the gas molecule concentration in solution is increased,and when the pressure is lowered, the excess gas is released as vapor.

The pressure-freeze method of certain embodiments of the presentinvention is based on this principle. An essential role of freezing isto concentrate both gas and solute molecules so as to favor theirencapsulation. Indeed, the basic phenomenon, that during freezing, airis released and often trapped as bubbles in the resultant ice, has beenknown for many years, and, moreover, that bubble formation in cellscontributes significantly to freezing damage in long-term preservationof cells and tissues.

Exemplary steps of the methods of producing echogenic liposomes areprovided below, particularly in Example 1 and FIGS. 1 and 7. Gasincorporation in liposomes is proportional to pressure. As noted above,this is to be expected from Henry's Law if gas uptake by the liposomesis proportional to the amount in solution at the first step. While thisinfluences gas entrapment, it is the freezing step that has a largeinfluence on dissolved gas and hence on gas encapsulation. While thepresent invention is not limited to any mechanism, it is believed thatfreezing probably serves two purposes, increasing the localconcentration of dissolved gas and nucleating formation of small pocketsof bulk gas phase. Gases, like other solutes, are more soluble in liquidwater than in solid ice. Thus, as the ice crystals grow, dissolved gasis progressively displaced from ice to unfrozen solution, with theresult that the dissolved gas becomes increasingly concentrated in theever-diminishing volume of liquid solution. When the dissolved gasconcentration becomes sufficiently high, a gas bubble may nucleate andgrow. According to the nucleation theory, bubbles form when thedifference between the total dissolved gas pressure and the ambientpressure in the surrounding liquid exceeds the Laplace pressure (thepressure created in a bubble due to the contraction of the surface underthe influence of the surface tension).

Although it is clear that freezing expels the gas from the aqueousphase, it is unknown where the bubbles so expelled reside within thefrozen dispersion. In order for the dispersion to becomeultrasound-reflective, there must be pockets of air with surfaces ofhigh acoustic impedance (as shown in FIG. 1). The gas might come out ofsolution in contact with the hydrophobic interior of the lipid bilayer,which has a relatively low surface tension against air; however, theeffect of trehalose on air incorporation suggests a more complex processis involved. Trehalose, which functions as a cryoprotectant by favoringglass formation rather than crystallization (either of itself or of thewater), supported much less echogenicity than did mannitol. Mannitol israther distinctive among sugars in readily crystallizing out of solutionupon cooling. Previously, it was proposed that freezing a mannitolsolution inflicts damage upon liposomes. Consistent with that findingand based on nucleation theory is a suggestion made a number of yearsago that the polar-nonpolar interface at the surface of damagedmembranes may be the preferred site of nucleation of release of nitrogenbubbles in decompression sickness which affects divers who are rapidlydecompressed.

Again, while the present invention is not limited to any particularmechanism, the following is believed to be part of the gas-containingliposome formation process. Although supersaturation of the liquid phaseduring ice formation should cause incipient air pockets to form, it isunlikely that this is the whole story, for, if it were, the echogencityshould not be particularly low when samples are thawed prior to reducingthe pressure back to ambient pressure. Under these conditions, the icemelts and the water produced is essentially degassed, so air associatedwith lipid (in all forms) will diffuse into this water. On the otherhand, when the pressure is lowered first and the sample thawed second,the air concentration in the solution that melts initially is highbecause it contains most of the air that dissolved in the suspensionupon pressurization. Because of its high solute (mannitol) content, theice in the environment of the liposomes will melt first and immediatelyexpose the lipid to ambient (1 atm) pressure. This initially meltingphase is not only highly supersaturated with air, but it is also likely,as described in the preceding paragraph, to contain air pockets thatwill grow when exposed to ambient pressure. Hence, air will come out ofsolution, expanding the gas nuclei that presumably formed duringfreezing. The result is the formation of air pockets that are stabilizedby a monolayer of lipid.

Furthermore co-encapsulation of gas (e.g., Xe) and an aqueous solute hasadvantages in drug delivery as it allows for release of liposomalcontents by application of ultrasound. Since acoustically activeliposomes also reflect ultrasound, it is possible to not only tolocalize the release of the drug according to the site of application ofultrasound, but also to image the therapeutic agent while it is beingactivated for delivery. Moreover, molecular targeting of the liposomesthemselves is possible.

In addition to releasing liposomal contents and providing an image ofthe process, ultrasound can have effects on the tissue that synergizewith drug delivery, namely cavitation effects of ultrasound which canfacilitate access of the drug to its target. For example, prior methodsfound site-specific drug delivery can be achieved by destroyingdrug-filled contrast microbubbles in the target area with high-intensityultra-sound (Porter et al., J Ultrasound Med 1996; 15(8):577-84.). Inaddition, Shohet et al. (Circulation 2000; 101(22):2554-6.) found thatalbumin-coated microbubbles could be used to effectively deliver anadenoviral transgene to rat myocardium via US-mediated microbubbledestruction. Prior work has also found enhanced uptake of plasmid DNA inthe presence of acoustically active liposomes and with the simultaneousapplication of ultrasound (Huang et al., Mol. Ther. 2003; 7 (5): 422Part 2).

The sensitivity of echogenic liposomes to ultrasound stimulation may beable to be improved further by varying the liposomal composition, theencapsulated gas and/or the ultrasound application parameters. The lipidbilayer is held together by hydrophobic interactions that tend to endowit with self-sealing properties such that the lipid shell of a liposomerapidly reseals following surface alternations. It is therefore probablethat changing the rigidity of the lipid membrane will affect itsresponse to ultrasound. The choice of the optimal gas will involve bothhigh volume in the liposomes and low rate of release in the bloodstream. The most effective ultrasound pulses would seem to be a smallnumber at the highest intensity that the tissue can sustain.

B. Targeting of Liposome

Targeted delivery is achieved by the addition of ligands withoutcompromising the ability of liposomes to deliver their payloads. It iscontemplated that this will enable delivery to specific cells, tissuesand organs (e.g., specific sites in the brain). The targetingspecificity of the ligand-based delivery systems is based on thedistribution of the ligand receptors on different cell types. Thetargeting ligand may either be non-covalently or covalently associatedwith a nanoparticle, and can be conjugated to the nanoparticles by avariety of methods as discussed herein.

Examples of molecules that could be used to target liposomes of theembodiments include antibodies (or fragments thereof) and aptamers.Alternatively or additional it is contemplated that cell-penetratingpeptides may be used to deliver liposomes directly into cells.

III. Pharmaceutical Compositions and Routes of Administration

Where clinical application of liposomes (e.g., liposomes comprisinggases) is undertaken, it will be necessary to prepare the liposomecomplex as a pharmaceutical composition appropriate for the intendedapplication. Generally, this will entail preparing a pharmaceuticalcomposition that is essentially free of pyrogens, as well as any otherimpurities that could be harmful to humans or animals. One also willgenerally desire to employ appropriate buffers to render the complexstable and allow for uptake by target cells. Aqueous compositions of thepresent invention comprise an effective amount of Xe encapsulated in aliposome as discussed above, further dispersed in pharmaceuticallyacceptable carrier or aqueous medium. Such compositions also arereferred to as inocula. The phrases “pharmaceutically” or“pharmacologically acceptable” refer to compositions that do not producean adverse, allergic or other untoward reaction when administered to ananimal, or a human, as appropriate. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients also can be incorporatedinto the compositions.

Solutions of therapeutic compositions can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersionsalso can be prepared in glycerol, liquid polyethylene glycols, mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The therapeutic compositions of the present inventionare advantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. These preparations also may be emulsified. A typicalcomposition for such purpose comprises a pharmaceutically acceptablecarrier. For instance, the composition may contain 10 mg, 25 mg, 50 mgor up to about 100 mg of human serum albumin per milliliter of phosphatebuffered saline. Other pharmaceutically acceptable carriers includeaqueous solutions, non-toxic excipients, including salts, preservatives,buffers and the like.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate.Aqueous carriers include water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial agents, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto well known parameters. Additional formulations are suitable for oraladministration. Oral formulations include such typical excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand the like. The compositions generally will take the form of solutionsor suspensions.

The therapeutic compositions of the present embodiments may includeclassic pharmaceutical preparations. Administration of therapeuticcompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. In thiscase, intravenous injection or infusion may be preferred. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions that include physiologically acceptablecarriers, buffers or other excipients.

An effective amount of the therapeutic composition is determined basedon the intended goal. The term “unit dose” or “dosage” refers tophysically discrete units suitable for use in a subject, each unitcontaining a predetermined-quantity of the therapeutic compositioncalculated to produce the desired responses, discussed above, inassociation with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the protection desired.

An effective dose range of a therapeutic can be extrapolated fromeffective doses determined in animal studies. In general a humanequivalent dose (HED) in mg/kg can be calculated in accordance with thefollowing formula (see, e.g., Reagan-Shaw et al., FASEB J.,22(3):659-661, 2008, which is incorporated herein by reference):

HED (mg/kg)=Animal dose (mg/kg)×(Animal K _(m)/Human K _(m))

Use of the K_(m) factors in conversion results in more accurate HEDvalues, which are based on body surface area (BSA) rather than only onbody mass. K_(m) values for humans and various animals are well known.For example, the K_(m) for an average 60 kg human (with a BSA of 1.6 m²)is 37, whereas a 20 kg child (BSA 0.8 m²) would have a K_(m) of 25.K_(m) for some relevant animal models are also well known, including:mice K_(m) of 3 (given a weight of 0.02 kg and BSA of 0.007); hamsterK_(m) of 5 (given a weight of 0.08 kg and BSA of 0.02); rat K_(m) of 6(given a weight of 0.15 kg and BSA of 0.025) and monkey K_(m) of 12(given a weight of 3 kg and BSA of 0.24).

Precise amounts of the therapeutic composition depend on the judgment ofthe practitioner and are peculiar to each individual. Nonetheless, acalculated HED dose provides a general guide. Other factors affectingthe dose include the physical and clinical state of the patient, theroute of administration, the intended goal of treatment and the potency,stability and toxicity of the particular therapeutic formulation. Forthe instant embodiments, it is envisioned that the amount of therapeuticliposome (e.g., Xe-ELIP) dose in a human (adult) will be greater thanabout 0.568 mg/kg. For example, the human dose range can be betweenabout 0.6 and 3.0 mg/kg, between about 0.8 and 2.8 mg/kg, or betweenabout 1.0 and 2.5 mg/kg. In some specific aspects Xe-ELIP isadministered to a human subject in a dose of between about 1.14 mg/kgand about 2.27 mg/kg.

IV. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Xe-ELIP Production and Experimental Methods

Xe-ELIP Production

Xenon-ELIP were produced using the freeze thaw protocol that isschematically illustrated in FIG. 7 (see also, U.S. Pat. No. 7,976,743,incorporated herein by reference). Briefly, liposomes were composed ofL-α-phosphatidylcholine (egg PC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; Avanti Polar Lipids,Alabaster, Ala.);1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (16:0 PEG2000 PE) and cholesterol (Sigma, St Louis, Mo.).Five milligrams of lipids was mixed in chloroform, and the solvent wasevaporated with argon in a 50° C. water bath to form a thin film on theglass vial. The lipid film was placed under vacuum for 4 to 6 hours forcomplete solvent removal. The dried lipid film was hydrated with 0.32mol/L mannitol to a concentration of 10 mg lipid per milliliter,followed by sonication for 5 minutes. The sonicated liposomes weretransferred to a 2-mL glass vial with a cap sealed with a Teflon-rubberseptum. Six milliliters of Xe (100%) (Concorde Specialty Gas Inc,Eatontown, N.J.) was injected into the glass vial through theTeflonrubber septum with a 12-mL syringe attached to a 27-gauge-½-inchneedle (note that at this stage other gases and/or gas mixtures may beincorporated). The pressurized liposomal dispersion was frozen at −70°C. with dry ice for at least half an hour. The liposomal dispersion wasallowed to thaw after the vial was unpressurized by removing the cap.The structure and gas retention properties of Xe-ELIP are shown in FIG.1.

Rat Model of MCA Occlusion

All animal experiments were approved by the Animal Welfare Committee atThe University of Texas Health Science Center at Houston. MaleSprague-Dawley rats (260-280 g, Harlan Laboratories Inc., Indianapolis,Ind.) were fasted for 24 hours with free access to water prior tosurgery. Before surgery, anesthesia was induced by placing rodent in asealed induction chamber (ask Melanie) for 5 minutes with a continuousflow of isoflorane. Marcaine (2 mg/kg) was injected subcutaneously atthe surgical site to provide topical analgesia. Cerebral ischemia wasinduced by occluding the right middle cerebral artery (MCA) for 2 hoursusing the intraluminal suture method described previously (Britton, etal. 2010, incorporated herein by reference). In brief, a 1 mm diameterburr hole was made in the skull to facilitate local cerebral perfusion(CP) measurement before occluding the MCA. Next, the right commoncarotid artery (CCA) was exposed through a midline neck incision. Theright external carotid artery (ECA) was then ligated close to its distalend. The internal artery was isolated and separated from adjacenttissues. A fabricated 25-cm 4-0 nylon monofilament was advanced from theright ECA and inserted into the right MCA for 2 hours to provokeischemia. Interruption of local blood flow through the MCA was verifiedwith a laser Doppler flowmeter placed over the ischemic area at 2 mmposterior and 6 mm lateral to the bregma. In all experiments, bodytemperature was maintained at 37° C. during ischemia. A polyethylenecatheter was introduced into the right femoral artery for pressurerecordings.

Determination of the Therapeutic Time Window

Animals were randomly divided into four groups (n=8 in each group), (1)no treatment group—MCA occlusion only; (2) treatment group “a”—Xe-ELIPadministration 10 min after reperfusion; (3) treatment group “b”—Xe-ELIPadministration 60 min after reperfusion; (4) treatment group “c”—Xe-ELIPadministration 180 min after reperfusion. All rats in each treatmentgroup were administrated 200 μl of Xe-ELIP over a period of 4 minutes bycannulating the right internal carotid artery with modified PESO tubing.The ICA was exposed to 1-MHz continuous wave ultrasound at apeak-to-peak pressure amplitude of 0.18 MPa (1-W/cm2 dial setting)during Xe-ELIP administration. Neurological assessments were conductedover the following three days. On the third day after MCAO the infarctvolume was determined by 2% 2,3,5-triphenyltetrazolium chloride (TTC)staining.

Determination of the Dose Dependence and Effect of Xe-EL IPAdministrations

Animals were randomly divided into four groups (n=8 in each group), (1)no treatment group—MCA occlusion only; (2) treatment group “a”—receiveda 100 μl dose of Xe (10 mg Xe-ELIP/ml); (3) treatment group “b”—receiveda 200 μl dose of Xe-ELIP (10 mg Xe-ELIP/ml); (3) treatment group “c”received a 400 μl dose of Xe-ELIP (10 mg Xe-ELIP/ml). All rats in eachtreatment groups received Xe-ELIP 60 min after reperfusion bycannulating the right internal carotid artery with modified PESO tubing.The ICA was exposed to 1-MHz continuous wave ultrasound at apeak-to-peak pressure amplitude of 0.18 MPa (1-W/cm2 dial setting)during Xe-ELIP administration. The infarct volume was determined 3 daysafter MCAO by TTC staining. Neurological assessments were conducted overthe following three days. On the third day after MCAO the infarct volumewas determined by 2% TTC staining.

Neurologic Assessment

All behavioral tests in mouse were conducted in a quiet and low-lit roomby an observer blinded with respect to the treatment groups. At days 1,2 and 3 after surgery, each animal was tested for motor function andneurologic outcomes by recording limb placing, beam walking and gridwalking abilities.

Limb placement was assessed by observing the animal's ability to liftits head and extend its forelimbs toward a table while the animal wassuspended over the table by its tail (zero score—no response; score of1-10 when response was sluggish or delayed; score of 2 when response wasrapid and fully executed). The ability to walk across a beam (2.5×2.5×80cm) was assessed by observing the ability to maintain balance whilenavigating across the beam. The response scores were assigned asfollows: score 0—traversed the beam with no foot slip; score 1—traversedwith grasping of the lateral side of the beam; score 2—showed difficultycrawling across the beam but able to traverse; score 3—required a morethan 10 seconds to traverse the beam due to difficulty in walking; score4—unable to traverse the beam; score 5—unable to move the body or anylimb on the beam; score 6—unable to stay on the beam for more than 10seconds. Grid walking ability was assessed by placing the animal on astainless steel grid floor (20 cm×40 cm with a mesh size of 2 cm×2 cm).The total number of steps was counted up to a maximum of 50 steps. Thenumber of foot fault errors as defined by the misplacement of a forelimbor hindlimb that fell through the grid was recorded.

Infarct Volume Measurement

Animals were sacrificed on the third day following neurologicalassessment. Brains were harvested. Using a Jacobowitz brain slicer, 2 mmthick coronal sections were cut prior to staining with 2%2,3,5-triphenyltetrazolium chloride (TTC) in PBS for 20 minutes at 3 rCfor infarct volume determination. Stained sections were transferred to10% phosphate buffered formalin for storage. Sections were photographedwith a Canon G7 10.0 megapixel camera fitted on a Polaroid land-tripodat an object distance of 8.5 cm. Images were transferred and analyzedwith Image Pro-Plus to calculate infarct volumes. Infarct volume wascalculated by measuring infarct areas on evenly sliced (1 mm) brainsections and adding them together (Simpson's rule). Normalized infarctvolume with respect to whole brain volume was calculated by dividing thevolume of TTC unstained (infarcted) tissue by that of the whole brain.

Gel Electrophoresis and Immunoblotting

Animals were subjected into three groups: 1) sham surgery withoutischemia; 2) MCAO for 2 hours without treatment; and 3) MCAO for 2 hoursand reperfusion for 1 hour following Xe-ELIP (400 μl) administrationintra-artery. Brain tissue slices were collected and homogenized in 1 mlof RIPA (Radio Immuno Precipitation Assay) buffer (Cell SignalingTechnology, MA, USA) containing the protease inhibitors,phenylmethylsulfonyl fluoride (PMSF, 1 mM), and phosphatase inhibitorcocktail (Santa Cruz Biotechnology, CA, USA). Brain tissues wereharvested at 7 and 24 hours after stroke onset. Whole cell protein wasextracted and sonicated with SONICS Vibra Cell (SONICS & MATERIALS Inc,CT, USA) for three times. The supernates were collected and proteinconcentration was measured. Equal amount of protein (80 μg) were loadedand separated on 12% SDS-Polyacrylamide gels with electrophoresis ofTris-glycine running buffer system for 2 hours, and then transferred toa polyvinylidene difluoride (PVDF) membrane (Millipore, Mass., USA).After blocking in Non Mammalian Blocking Reagent (LI-COR Biosciences,NE, USA) without tween-20 for 1 hour, the membranes were incubated withprimary antibodies of BDNF (1:250, Santa Cruz Biotechnology, CA, USA),phosphorylated AKT (1:250, Cell Signaling Technology, MA, USA) or totalAkt (1:500, Cell Signaling Technology, MA, USA), and Phospho-ERK1/2(1:250, Cell Signaling Technology, MA, USA) or Erk1/2 (:500, CellSignaling Technology, MA, USA) at 4° C. overnight. After washingmembranes with Tris-buffered saline containing 0.1% Tween-20 (TBS-T),the membrane was incubated with IRDye 800CW Dky Anti-Rabbit IgGsecondary antibody (H+L) (LI-COR Biosciences, NE, USA) at roomtemperature for 1 hour. After extensive washes (Rinse twice and wash 3times×5 min) in TBS-T 0.1%, blots were visualized by odyssey infraredimaging system (LI-COR Biosciences, NE, USA). To ensure equivalentprotein loading, membranes were reprobed with Anti b-actin (Sigma, MO,USA) for 1 hour at room temperature, and then incubated with IRDye 680 LT Gt Anti-Mouse IgG (H+L) (LI-COR Biosciences, NE, USA) for 1 hour.Membranes were scanned using same odyssey infrared imaging system. Theoptical densities of all protein bands were analyzed by using NIH ImageJsoftware. All target proteins were quantified by normalizing to b-actinand calculated as fold of the corresponding control group.

Statistical Analyses

Nonparametric statistical analyses were performed by the Wilcoxon ranktest for two groups or Kruskal-Wallis analysis of variance (ANOVA) formultiple groups, and reported as mean and standard deviation for mostexperiments. When differences were detected in global comparison, themultiple comparisons of mean ranks for all groups were performed for allpairwise comparisons. Neurologic outcome comparison between thetreatment groups was reported as median and quartiles. Statistica(Version 9, StatSoft Inc., Tulsa, Okla.) software were utilized forstatistical analyses. A p<0.05 was considered significant.

Example 2 Xe-ELIP as Therapeutic for Stroke

The dose dependency Xe-ELIP therapy was first investigated by injectingXe-ELIP at dosage range of 1-4 mg/rat (3.5, 7 and 14 mg/kg) at 3 hoursafter stroke onset on a Male Sprague-Dawley rat model with occlusion(for 2 hours) of the right transient endovascular filament middlecerebral artery (MCAO). Treatment groups that received 7 mg/kg or 14mg/kg of Xe-ELIP at 3 hours after stroke onset reduced the normalizedinfarct size to 6.0±2% (p=0.04) and 3.7±2% (p=0.002) respectively (FIG.4 a-e). This study demonstrates that Xe-ELIP administered within 3 hoursafter stroke onset at a dosage larger than 2-4 mg (e.g., 7 mg/kg orgreater) provide best neuroprotection.

Behavioral assessments of neurological damage were conducted byrecording limb placing, beam walking and grid walking abilities in aquiet and low-lit room in an observer blinded manner at days 1, 2 and 3after surgery. Results are shown in FIG. 4 f-h demonstrate verysignificant behavioral improvement in animals treated with 7 mg/kg or 14mg/kg.

The therapeutic time window of Xe-ELIP was then further investigated onboth rat filament MCAO and thrombotic MCAO model. Xe-ELIP wasadministrated through the ascending right common carotid artery at 2, 3,and 5 hour after stroke onset (10 min, 1 hour and 3 hour afterreperfusion) on filament MCAO model. In the non-treatment group, a largeinfarction developed and predominantly involved the cerebral cortex andstriatum with normalized infarct volume of 16±5.2% (228±74 mm³) of wholebrain (FIG. 2 a, e). Xe-ELIP administration at 10 min and 2 hours afterstroke onset reduced the normalized infract size from 15±5.1% (control)to 4.9±1.2% (p=0.005) and 6.0±3.4% (p=0.002), respectively (FIG. 2 a-e).There was no difference in core body temperature between the groupsduring MCA occlusion and the initial hours of reperfusion.

Behavioral assessment of neurological damage were conducted by recordinglimb placing, beam walking and grid walking abilities in a quiet andlow-lit room in an observer blinded manner at days 1, 2 and 3 aftersurgery. The group with Xe-ELIP administered at 3 hours after stroke onset demonstrated marginal improvements in performing behavioral tasks.Both Xe-ELIP administered at 10 minutes and 1 hour groups demonstratedimproved performance in all behavioral tests from day 1 with markedimprovements in all tests by day 3 (FIG. 2 f-h). It was demonstratedthat Xenon protected neuron damage as a glutamate receptors (NMDA)antagonist while the excitotoxic effect caused by excessiveextracellular glutamate accumulation was absorbed in an early event ofischemia.

The therapeutic time window of Xe-ELIP at 2 hour after stroke onset (10min after reperfusion) showed therapeutic effect. In the clinicalsetting, the tPA administration for stroke treatment is limited by itsnarrow therapeutic time window. Although 85% of strokes are due toocclusion of cerebral artery by a circulating clot, 15% of strokes arehemorrhagic. IV tPA cannot be administrated until the exclusion ofhemorrhage stroke from thrombotic stroke. Thus, neuroprotective agentadministration before IV tPA to prolong tPA therapeutic time window is avery promising clinical relevant strategy. Thus the neuroprotectiveeffect of Xe-ELIP administration was further investigated at 2 hourafter stroke onset but before and after reperfusion. The reduction ofbrain infarct by Xe-ELIP before and after treatment is shown in FIG. 6a. Xe-ELIP before reperfusion and after reperfusion reduced thenormalized infract size by 86±12% and 67±7%, respectively.

The administration of tPA within 4.5 h of ischemic stroke onset remainsthe only treatments that have been shown to have clinical benefit.Neuroprotective combination therapy may minimize the harmful effects ofischemic neuronal damage. To test the effect of Xe-ELIP on tPA activity,thrombolytic efficiency of tPA in the present of Xe-ELIP was comparedwith tPA alone on porcine blood clot. The thrombolytic effect of tPA wasinhibited after 30 minute incorporation with free Xe (Xe saturatedsolution). When tPA was incorporated with Xe-ELIP, it had the samethrombotic effect as tPA alone. This demonstrated a protective effect ofELIP on Xe from interaction with tPA (FIG. 6 b).

The therapeutic effect of Xe-ELIP in a rat embolic stroke model was nextinvestigated. Thrombotic strokes were induced in male Sprague-Dawleyrats (n=16) by injecting a 13 mm long blood clot into the middlecerebral artery. In the treatment group, tPA (10 mg/kg) was infusedintravenously at 2 hours after the onset occlusion. Xe-ELIP wasadministrated intravenously before IV tPA. Continuous wave ultrasound (1MHz, 50% duty cycle, 0.5 W/cm²) was applied to trigger Xe release fromELIP during the 5 min of Xe-ELIP administration. The thrombotic strokecontrol group without any treatment exhibited the largest damage andinfarct size (17±5% of the whole brain) (FIG. 5 a-e). The tPA treatmentreduced the damage and the infarct size to 5.2±0.4% (p=0.025 vs.stroke). The tPA treatment in combination with Xe-ELIP further reducedinfarct size to 1.5±0.4% (p=0.05 vs. tPA group). Behavioral deficitcorrelated inversely with infarct volume. Regional blood flow velocitymonitored by a laser Doppler flow meter was similar in both tPA andtPA+Xe-ELIP treatment groups (FIG. 5 f-h). This study demonstrated aneuroprotective effect of ELIP encapsulated xenon released byapplication of 1 MHz ultrasound. Xe-ELIP can be used in combination withtPA without affecting tPA thrombolytic activity. Death rates resultingfrom the treatment conditions are shown in Table 1 below.

TABLE 1 Reperfusion rate Group Death rate Occlusion rate after IV tPASham 0 0 — Stroke 60% 58 ± 9% — Stroke + tPA 31% 50 ± 8% 21 ± 17%Stroke + Xe-ELIP + 29% 53 ± 6% 23 ± 14% tPA

The effects of Xe-ELIP on BDNF expression and apoptosis was alsoassessed (see, e.g., FIGS. 3). Western blot analysis of BDNF (a),phos-Akt (b) and phos-ERK (c) in cerebral cortex tissue 24 h afterstroke showed that Xe increased the expression of BDNF (d), total Akt(e) and phos-ERK (f) (FIG. 3 a-f). TUNEL staining in the penumbralregion of brain sections from the sham-operated group (g), stroke group(h) and stroke with Xe-ELIP treatment group (i) showed reduction ofapoptosis in Xe-ELIP-treated animals (FIG. 3). The Western blots andphotomicrographs of apoptosis are representative of three independentexperiments. Data are means±SD.

Example 3 Xe-ELIP Provides Effective Protection in Hemorrhagic Stroke

Xe-ELIP compositions were produced as detailed in Example 1. To assessthe efficacy Xe-ELIP a rat model of subarachnoid hemorrhage wasemployed. Briefly, healthy male Sprague Dawley rats (Harlan LaboratoriesInc., Indianapolis, Ind.) weighing between 260-280 grams were obtained.All surgical procedures were performed under dissecting microscope onanesthetized animal. The right external carotid artery was isolated anda 4.0 fabricated sharp nylon monofilament was introduced through theinternal artery to perforate the middle cerebral artery. Themonofilament was immediately retracted to resume blood flow to themiddle cerebral artery. The blood flow was monitored to confirm thebleeding.

Following the induction of bleeding Xe-ELIP (600 μl, 10 mg/ml) wasinfused for 5 minutes through the femoral vein with simultaneousultrasound application (0.5 MPa) over the internal carotid artery totrigger the release of xenon from circulating Xe-ELIP into brain.Neurological and behavioral tests were conducted for 3 days followingsurgery. Animals were sacrificed for SAH grading to evaluate the degreeof bleeding, brain water contain to evaluate the edema and TUNELstaining to check the apoptosis, on the third day following neurologicaland behavior assessments.

Results from the physical examination of brain tissue are shown in FIG.8. For scoring the basal cistern was divided into six segments. Bleedingwas assessed in each of these segments and scored from 0 to 3 (0: noSAH; 1: minimal SAH; 2: moderate blood clot with recognizable arteries;3: blood clot obliterate the arteries). The total scores were added andthe severity of bleeding was scaled as: 0-7: mild SAH; 8-12: moderateSAH; 13-18: severe SAH.Xe-ELIP decreases the bleeding in filamentperforation subarachnoid hemorrhage (SAH) rat model

Results of behavioral testing of the treated rats are shown in FIG. 9.Lower panel illustrates the array of behavioral tests to which animalswere subject. Results of neurological evaluation, beam walking and gridwalking are shown in the graphs of the upper panel. In each case ratstreated with Xe-ELIP performed significantly better than untreated rats.Indeed, microscopic examination of brain sections using TUNEL staining(FIG. 10) showed that brain sections from hemorrhage stroke group, upperpanels, had significantly more apoptotic cells as compared to thehemorrhage stroke with Xe-ELIP treatment group, bottom (compare centerpanels). Perhaps most importantly, Xe-ELIP treatment decreased the deathrate of SAH rats but did not show significant effects on brain edema andcerebral blood flow (FIG. 11). Brain edema is the major life-threateningcomplication of stroke. It is frequently associated with subarachnoidhemorrhage, vasospasm and ischemic reperfusion damage. The results shownhere demonstrate that ELIP formulations containing only one gas (Xe)does not affect the brain edema and vasoactivity. It has been shown thathydrogen administration after stroke can eliminate the brain edema bydecreasing blood-brain barrier permeability, and H₂S inhibits vasospasmby anti-inflammatory effect. Thus, in some aspects, formulations thatco-encapsulate hydrogen gas and/or hydrogen sulfide gas with Xe-ELIPwould have added effect on brain edema and cerebral vasospasm.

Example 4 H₂ and H₂S Enhance Xe-ELIP Efficacy

Studies were next undertaken to evaluate co-encapsulation of Xe withhydrogen or hydrogen sulfide into ELIP. ELIP were composed ofphospholipids and cholesterol and were produced as detailed inExample 1. In this case, however, gas mixtures of 30% hydrogen+70% xenonor 1% hydrogen sulfide+99% xenon were loaded onto ELIP by thepressurized-freeze method in addition to use of 100% Xe.

The design of efficacy experiments is graphically represented in FIG.12. As indicated, in order to test the therapeutic effect of H₂/Xe-ELIPor H₂S/Xenon ELIP, 400 μl of each (in additional Xe-ELIP alone) wereadministered into Sprague-Dawley rats intravenously separately after (at3 h) right middle cerebral artery occlusion. One-megahertz low-amplitude(0.18 MPa) continuous wave ultrasound directed onto the internal carotidartery was used to trigger gas release from circulating Xe-ELIP.

Animals were then subjected to behavioral testing and their brainsexamined to assess the physical damage present. As shown in FIGS. 13 aand b, both addition of H₂ and H₂S further increased the ability ofXe-ELIP to reduce the normalized infarct volume in the brains of treatedrats. As shown in FIG. 13 c, addition of H₂S further increased theability of Xe-ELIP to reduce the number of TUNEL positive cells in thebrains of treated rats, indicated reduce neuron cell damage. Analysis ofneutrophil invasion across the vascular wall in the brains of treatedrats showed decreased invasion following Xe-ELIP treatment, which wasenhanced by H₂S. Inhibition of neutrophil transfer across the vascularwall is one potential mechanism for H₂S/Xe-ELIP neurovascular unitprotection.

Perhaps more importantly animals treated Xe-ELIP combined with of H₂ orH₂S also tended to perform better in behavioral testing that includedlimb placement, beam walking and grid walking (FIG. 14). In particular,the combined therapy was show to be significantly better in improvementof grid walking ability as compared to both control (untreated) rats andrats treated with Xe-ELIP alone.

Example 5 Xe-ELIP Protection of Culture Human Brain Astrocytes AgainstHydrogen Peroxide (H₂O₂) Cytotoxicity or Oxidative Stress

Human brain astrocytes play a key role in maintaining nerve cellfunction and survival against oxidative stress. Exposure of culturedhuman brain astrocytes to H₂O₂ causes significant damages to the cells,and caused them to release large amounts of LDH. However, pretreatmentof the brain cells with Xe-ELIP reagents markedly reduced LDH release(FIG. 15), indicating a protective effect of Xe-ELIP on the brain cellsinjured by the oxidative stress. No or little protective effect wasfound in the cells treated with ELIP alone or control media (FIG. 15).

Example 6 Xe-ELIP has No Cytotoxicity to Murine Stem Cells

Murine embryonic stem cells were examined for their growth and survivalwhen they were treated with or without H₂O₂ in the presence or absenceof Xe-ELIP. The cell viability was determined by assessing the releaseof LDH. In the presence of ELIP loaded with or without Xe or othergases, LDH levels in the cultures remained significant levels (FIG. 16).By contrast, addition of H₂O₂ (10 mM), significant LDH release was foundwithin 2 hours of exposure to the oxidative stress agent H₂O₂ (FIG. 16).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of treating a hemorrhagic stroke in asubject comprising administering an effective amount of a compositioncomprising xenon-loaded echogenic liposomes to the subject.
 2. Themethod of claim 1, wherein the composition is administeredintravenously, intra-arterially, intracranially, via intravenousinfusion or via intra-arterial infusion.
 3. The method of claim 1,further comprising applying ultrasound stimulation to the subject in anamount effective to promote xenon release from the liposomes.
 4. Themethod of claim 3, wherein the ultrasound stimulation is applied with aconventional ultrasound probe or a cervical collar ultrasound device. 5.The method of claim 3, wherein the ultrasound stimulation is applied tothe neck or head of the subject.
 6. The method of claim 3, wherein theultrasound stimulation is applied at a frequency of between about 1 and8 MHz and with a mechanical index of between about 0.1 and 1.4.
 7. Themethod of claim 1, wherein the administration is within 6 hours ofstroke onset.
 8. The method of claim 1, wherein the administration iswithin 4, 3, 2 or 1 hour of stroke onset.
 9. The method of claim 1,wherein the subject is human.
 10. The method of claim 1, wherein thecomposition is administered to the subject a second time.
 11. The methodof claim 10, wherein the second administration is about 2, 3, 4, 5, 6,7, or 8 hours after the initial administration.
 12. The method of claim1, further comprising administering a second therapeutic agent to thesubject.
 13. The method of claim 12, wherein the second therapeuticagent comprises H₂S or H₂-loaded echogenic liposomes.
 14. The method ofclaim 13, wherein the H₂S or H₂-loaded echogenic liposomes and theXe-loaded echogenic liposomes are comprised in the same composition. 15.The method of claim 14, wherein the composition comprises liposomesloaded with two gases.
 16. The method of claim 1, wherein administeringthe composition comprises suspending lyophilized liposomes in a solutionand administering the suspension to the subject.
 17. The method of claim1, wherein administering the composition comprises thawing a frozenliposome suspension and administering the suspension to the subject. 18.The method of claim 1, wherein the composition further comprises acryoprotectant.
 19. The method of claim 18, wherein the cryoprotectantis mannitol.
 20. The method of claim 1, wherein the liposomes comprise aphosphatidylcholine (PC), a phosphoethanolamine (PE), polyethyleneglycol (PEG), a phosphatidylglycerol (PG) or a phosphatidylserine (PS)or cholesterol.
 21. The method of claim 20, wherein the liposomescomprise at least one PC, PE, negatively-charged lipid, PEGylated lipidand cholesterol molecule.
 22. The method of claim 20, wherein: (a) thePC comprises dipalmitoyl phosphatidylcholine (DPPC) or EggPhosphatidylcholine (EPC); (b) the PEG comprises PEG2000-DPPE; or (c)the PG comprises 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol).23. The method of claim 22, wherein the liposomes comprise DipalmitoylPhosphatidylcholine (DPPC), Egg Phosphatidylcholine (EPC), PEG2000-DPPE,DPPG and cholesterol.
 24. The method of claim 23, wherein the liposomesconsist essentially of DPPC, EPC, PEG2000-DPPE, DPPG, cholesterol andxenon.
 25. The method of claim 1, wherein the liposomes have an averagesize of 0.4 to 10 microns.
 26. The method of claim 1, wherein thesubject has been diagnosed with a hemorrhagic stroke.
 27. A method oftreating stroke in a subject who has not been determined to have athrombotic or a hemorrhagic stroke comprising administering an effectiveamount of a composition comprising xenon-loaded echogenic liposomes tothe subject.
 28. The method of claim 27, wherein the subject has notbeen determined to have a thrombotic stroke.
 29. The method of claim 27,wherein the composition is administered intravenously, intra-arterially,intracranially, via intravenous infusion or via intra-arterial infusion.30. The method of claim 27, further comprising applying ultrasoundstimulation to the subject in an amount effective to promote gas releasefrom the liposomes.
 31. The method of claim 30, wherein the ultrasoundstimulation is applied with a conventional ultrasound probe or acervical collar ultrasound device.
 32. The method of claim 30, whereinthe ultrasound stimulation is applied to the neck or head of thesubject.
 33. The method of claim 30, wherein the ultrasound stimulationis applied at a frequency of between about 1 and 8 MHz and with amechanical index of between about 0.1 and 1.4.
 34. The method of claim27, wherein the administration is within 6 hours of stroke onset. 35.The method of claim 27, wherein the administration is within 4, 3, 2 or1 hour of stroke onset.
 36. The method of claim 27, wherein the subjectis human.
 37. The method of claim 27, wherein the composition isadministered to the subject a second time.
 38. The method of claim 37,wherein the second administration is about 2, 3, 4, 5, 6, 7, or 8 hoursafter the initial administration.
 39. The method of claim 27, furthercomprising administering a second therapeutic agent to the subject. 40.The method of claim 39, wherein the second therapeutic agent comprisesH₂S or H₂-loaded echogenic liposomes.
 41. The method of claim 40,wherein the H₂S or H₂-loaded echogenic liposomes and the Xe-loadedechogenic liposomes are comprised in the same composition.
 42. Themethod of claim 27, wherein administering the composition comprisessuspending lyophilized liposomes in a solution and administering thesolution to the subject.
 43. The method of claim 27, whereinadministering the composition comprises thawing a frozen liposomesuspension and administering the suspension to the subject.
 44. Themethod of claim 27, wherein the composition further comprises acryoprotectant.
 45. The method of claim 44, wherein the cryoprotectantis mannitol.
 46. The method of claim 27, wherein the liposomes comprisea phosphatidylcholine (PC), a phosphoethanolamine (PE), polyethyleneglycol (PEG), a phosphatidylglycerol (PG) or a phosphatidylserine (PS)or cholesterol.
 47. The method of claim 46, wherein the liposomescomprise at least one PC, PE, negatively-charged lipid, PEGylated lipidand cholesterol molecule.
 48. The method of claim 46, wherein: (a) thePC comprises dipalmitoyl phosphatidylcholine (DPPC) or EggPhosphatidylcholine (EPC); (b) the PEG comprises PEG2000-DPPE; or (c)the PG comprises 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol).49. The method of claim 48, wherein the liposomes comprise DipalmitoylPhosphatidylcholine (DPPC), Egg Phosphatidylcholine (EPC), PEG2000-DPPE,DPPG and cholesterol.
 50. The method of claim 49, wherein the liposomesconsist essentially of DPPC, EPC, PEG2000-DPPE, DPPG, cholesterol andxenon.
 51. The method of claim 27, wherein the liposomes have an averagesize of 0.4 to 10 microns.
 52. A method of treating both hemorrhagic andthrombotic stroke in a subject comprising administering an effectiveamount of a composition comprising xenon-loaded echogenic liposomes tothe subject.
 53. The method of claim 52, wherein the subject has beendiagnosed with a hemorrhagic and thrombotic stroke.
 54. The method ofclaim 52, wherein it has not been determined whether the subject ishaving a hemorrhagic or thrombotic stroke.
 55. A method of treatingstroke in a subject comprising: (a) identifying a subject having astroke who has not been determined to have a thrombotic or a hemorrhagicstroke; and (b) administering an effective amount of a compositioncomprising xenon-loaded echogenic liposomes to the subject.
 56. A methodof treating a thrombotic stroke in a subject comprising: (a)administering an effective amount of a first composition comprisingxenon-loaded echogenic liposomes to the subject; and (b) administeringan effective amount of a second composition comprising tissueplasminogen activator (tPA) to the subject.
 57. The method of claim 56,wherein the tPA is comprised in a liposome.
 58. The method of claim 56,wherein the tPA is recombinant human tPA.
 59. The method of claim 56,wherein the second composition further comprises xenon-loaded echogenicliposomes.
 60. The method of claim 56, wherein the first or secondcomposition is administered intravenously, intra-arterially,intracranially, via intravenous infusion or via intra-arterial infusion.61. The method of claim 56, further comprising applying ultrasoundstimulation to the subject after said first composition is administeredin an amount effective to promote gas release from the liposomes. 62.The method of claim 61, wherein the ultrasound stimulation is appliedwith a conventional ultrasound probe or a cervical collar ultrasounddevice.
 63. The method of claim 61, wherein the ultrasound stimulationis applied to the neck or head of the subject.
 64. The method of claim56, wherein the ultrasound stimulation is applied at a frequency ofbetween about 1 and 8 MHz and with a mechanical index of between about0.1 and 1.4.
 65. The method of claim 56, wherein the administration ofthe first composition is within 6 hours of stroke onset.
 66. The methodof claim 56, wherein the administration of the first composition iswithin 4, 3, 2 or 1 hour of stroke onset.
 67. The method of claim 56,wherein the subject is human.
 68. The method of claim 56, wherein theadministration of the second composition is administered about 2, 3, 4,5, 6, 7, or 8 hours after administration of the first composition. 69.The method of claim 56, wherein the first or second composition comprisea further therapeutic agent.
 70. The method of claim 69, wherein thefurther therapeutic agent comprises H₂S or H₂-loaded echogenicliposomes.
 71. The method of claim 56, wherein administering the firstcomposition comprises suspending lyophilized liposomes in a solution andadministering said solution to the subject.
 72. The method of claim 56,wherein administering the first composition comprises thawing a frozenliposome suspension and administering the suspension to the subject. 73.The method of claim 56, wherein the first or second composition furthercomprises a cryoprotectant.
 74. The method of claim 73, wherein thecryoprotectant is mannitol.
 75. The method of claim 56, wherein theliposomes of the first composition comprise a phosphatidylcholine (PC),a phosphoethanolamine (PE), polyethylene glycol (PEG), aphosphatidylglycerol (PG) or a phosphatidylserine (PS) or cholesterol.76. The method of claim 75, wherein the liposomes comprise at least onePC, PE, negatively-charged lipid, PEGylated lipid and cholesterolmolecule.
 77. The method of claim 75, wherein: (a) the PC comprisesdipalmitoyl phosphatidylcholine (DPPC) or Egg Phosphatidylcholine (EPC);(b) the PEG comprises PEG2000-DPPE; or (c) the PG comprises1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol).
 78. The methodof claim 77, wherein the liposomes comprise DipalmitoylPhosphatidylcholine (DPPC), Egg Phosphatidylcholine (EPC), PEG2000-DPPE,DPPG and cholesterol.
 79. The method of claim 78, wherein the liposomesconsist essentially of DPPC, EPC, PEG2000-DPPE, DPPG, cholesterol andxenon.
 80. The method of claim 56, wherein the liposomes of the firstcomposition have an average size of 0.8 to 10 microns.
 81. The method ofclaim 56, wherein the subject has been diagnosed with a thromboticstroke.
 82. A pharmaceutical composition comprising xenon-loadedechogenic liposomes and tPA.
 83. The composition of claim 82, whereinthe tPA is comprised in a liposome.
 84. The composition of claim 82,wherein the tPA is recombinant human tPA.
 85. A pharmaceuticalcomposition comprising Xe-loaded echogenic liposomes and liposomesloaded with H₂S or H₂.
 86. The composition of claim 85, wherein thegases are comprised in separate liposomes in the composition.
 87. Thecomposition of claim 85, wherein the gases are comprised in the sameliposomes.
 88. The composition of claim 85, comprising liposomes loadedwith Xe, H₂S and H₂.
 89. The composition of claim 82 or 85, wherein theliposomes comprise a phosphatidylcholine (PC), a phosphoethanolamine(PE), polyethylene glycol (PEG), a phosphatidylglycerol (PG) or aphosphatidylserine (PS) or cholesterol.
 90. The composition of claim 89,wherein the liposomes comprise at least one PC, PE, negatively-chargedlipid, PEGylated lipid and cholesterol molecule.
 91. The composition ofclaim 90, wherein: (a) the PC comprises dipalmitoyl phosphatidylcholine(DPPC) or Egg Phosphatidylcholine (EPC); (b) the PEG comprisesPEG2000-DPPE; or (c) the PG comprises1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol).
 92. Thecomposition of claim 91, wherein the liposomes comprise DipalmitoylPhosphatidylcholine (DPPC), Egg Phosphatidylcholine (EPC), PEG2000-DPPE,DPPG and cholesterol.
 93. The composition of claim 92, wherein theliposomes consist essentially of DPPC, EPC, PEG2000-DPPE, DPPG,cholesterol and xenon.
 94. A pharmaceutical composition comprisingXe-loaded echogenic liposomes wherein the said liposomes are comprisedof (1) a first PC component, (2) a second PC component, (3) a PEGylatedlipid component, (4) a PG component and (5) a cholesterol component andwhere in said components are present ratios of about30-60:10-30:5-15:5-15:10-20, respectively.
 95. The composition of claim94, wherein said components are present in ratios of about40-50:20-30:5-10:5-10:10-20, respectively.
 96. The composition of claim95, wherein: (a) the first PC component is dipalmitoylphosphatidylcholine (DPPC); (b) the second PC component is EggPhosphatidylcholine (EPC); (c) the PEGylated lipid component isPEG2000-DPPE; or (d) the PG component is1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol).
 97. Thecomposition of any of claim 82, 85 or 94, wherein the compositionfurther comprises a cryoprotectant.
 98. The composition of claim 97,wherein the cryoprotectant is mannitol.
 99. The composition of any ofclaim 82, 85 or 94, wherein the liposomes have an average size of 0.4 to10 microns.
 100. The composition of any of claim 82, 85 or 94, whereinthe composition is lyophilized or frozen.